Title

Author

Access Type

Open Access Dissertation

Date of Award

January 2011

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Pathology

First Advisor

Fazlul H. Sarkar

Abstract

The American Cancer Society estimated that 222,520 Americans were diagnosed with lung cancer and 157,300 died of lung cancer in 2010 (Jemal et al. 2009, 225-249;Jemal et al. 2011, 69-90). The clinical outcome of patients diagnosed with non-small cell lung cancer (NSCLC), the major lung cancer sub-types, is very poor, which calls for innovative research for finding novel therapeutic targets and agents for better treatment outcome.

Emerging evidences have suggested that a phenomenon called Epithelial-to-Mesenchymal Transition (EMT), which shares similar molecular characteristics with cancer stem-like cells, contributes to lung cancer treatment failure. In view of the fact that EMT process has been implicated in the two important biological processes that are accountable for cancer-related deaths; the progression of cancer cells to a distant organ, and the acquisition of resistance to conventional cancer therapeutics (Leng et al. 2011, 145-155;Shih and Yang 2011;Zhang et al. 2011), needs further in-depth investigation. Therefore, further mechanistic understanding of the role of EMT in lung cancer is very important, which was the focus of my investigation.

In this study, I found for the first time that the induction of EMT by chronic exposure of A549 NSCLC cells to TGF-Β1 (A549-M cells) led to the up-regulation of sonic hedgehog (Shh) both at the mRNA and protein levels causing activation of hedgehog signaling. These results were also reproduced in another NSCLC cell line (H2030), and these results were further associated with the induction of EMT phenotype. Induction of EMT was found to be consistent with aggressive characteristics such as increased clonogenic growth, cell motility and invasion. The aggressiveness of these cells was attenuated by the treatment of A549-M cells with pharmacological inhibitors of Hh signaling in addition to Shh knock-down by siRNA. The inhibition of Hh signaling by pharmacological inhibitors led to the reversal of EMT phenotype as confirmed by the reduction of mesenchymal markers such as ZEB1 and Fibronectin, and induction of epithelial marker E-cadherin. In addition, knock-down of Shh by siRNA significantly attenuated EMT induction by TGF-Β1.

Next, I examined the involvement of NF-κB, as downstream of TGF-Β-receptor signaling, and in the up-regulation of Shh. I tested whether NF-κB activity could indeed be induced by TGF-Β1 in our model cell culture system. To gain further mechanistic insight, I also tested whether the active NF-κB could bind to consensus sequences on Shh promoter in cell free system. Additionally, I determined the binding of the active NF-κB to Shh promoter in the cell by CHIP assay. Finally, I evaluated whether NF-κB can activate Shh expression directly using Shh promoter-luciferase reporter assays. The results clearly showed that TGF-Β1 induced NF-κB activity in NSCLC cell line A549. In addition, active NF-κB bound to its consensus sequences in the Shh promoter. Likewise, on CHIP assay, I found that active NF-κB bound to Shh promoter. Also, by utilizing promoter-luceferase reporter assays, I confirmed that upon TGF-Β1 treatment NF-κB was physically bound to Shh promoter, and activated its transcription and expression. Furthermore, I identified Shh gene as a target for miR15a and miR-16, the two miRNAs that has been reported to be deleted or down-regulated in NSCLCs.

To verify whether pharmacological inhibitors of Hh signaling pathway can sensitize mesenchymal tumors with resistant phenotype to standard therapy, and whether the sensitization of this tumor is a consequence of EMT reversal caused by the inhibition of hh signaling. For such studies, I treated NSCLCs A549-M, H1299, and H1650 cells with Hh inhibitors GDC-0449 for three days, and then further treated these cells with either cisplatin or erlotinib for different time points. My data showed an increase in NSCLCs sensitivity towards cisplatin and erlotinib, suggesting that Hh inhibitors sensitized drug resistant mesenchymal phenotypic NSCLC tumor cells to standard therapy by reverting EMT phenotype.

From these results, I conclude that chronic exposure of cancer cell in vivo to TGF-Β1 leads to the acquisition of EMT phenotype as documented by this in vitro study using NSCLC cells. The induction of EMT mediated by TGF-Β1 was in part due to transcriptional activation of Shh which was due in part resulting from the activation of NF-κB and SMAD signaling in NSCLC cells, and causing tumor cell aggressiveness in vitro and in animal model in vivo. The acquisition of EMT resulted in therapeutic resistance and the treatment of these cells with Hh signaling inhibitors (either Shh siRNA or synthetic inhibitor, GDC-0449) caused reversal of EMT and sensitized cells to conventional therapeutics. Together, these results suggest that reversal of EMT by Hh inhibitors would be useful for achieving better treatment outcome in patients diagnosed with NSCLC.